The M-type voltage-gated potassium current regulates action potential threshold and spike frequency adaptation. The M current is suppressed by various neurotransmitters including acetylcholine, which elicits hyperexcitable periods upon stimulation. To date, a number of mutations in the genes encoding the M channel, KCNQ2, 3, 4 and 5, have been reported to cause neurological disorders such as epilepsy and neuromyotonia. Accordingly, M channel modulators are considered potential therapeutic agents to control neuronal excitability in pathogenic conditions such as epilepsy, pain and cognition. The long-term goal of this project is to elucidate regulation and physiological relevance of the M current modulation as a model for understanding roles of low threshold voltage-gated channels in higher brain function. Several parallel regulatory pathways have been identified for mediating the neurotransmitter-induced suppression of the M channel, one of which is depletion of PIP2 by activation of phospholipase C. Accumulating evidence shows PIP2 is an essential cofactor for a wide variety of ion channels and transporters. This general requirement for PIP2 raises the question of how PIP2 deletion selectively regulates the M channel. The hypothesis addressed in this proposal is that the M channel reduces its affinity to PIP2 due to rearrangement of the macromolecular channel complex during the neurotransmitter-induced suppression. The specific aims are to: 1) link change in components of the M channel complex and reduction of PIP2 affinity during muscarinic cholinergic stimulation; 2) elucidate changes in the M channel complex induced by calcium and cross talks with other pathways. These aims will be tested by a combination of electrophysiological, biochemical and imaging approaches designed to address how the channel complex is arranged. Channel activity and conformational change will be recorded simultaneously using a patch clamp technique under FRET microscopy in transfected cultured cell lines and cultured neurons. This work will advance our understanding of how the M channel is regulated to control neuronal excitability.